Our preliminary data demonstrate that during tolerization there is alloantigen specific clustering of T cells and plasmacytoid DC (pDC) in the T cell areas of the lymph node (LN) near the abluminal surface of the high endothelial venules (HEV). Within these clusters T cells undergo priming or development into de novo CD4+CD25+ regulatory T cells (Treg), B cells present specific alloantigen in these cell clusters, and LN stromal cells structures are morphologically altered in rejection versus tolerization. We hypothesize these multicellular clustered interactions in the LN are key to the induction and maintenance of tolerance. Specifically, the interaction of T-APC (pDC, B cells) along with other cells and structures in LNs determines T cell migration, positioning, proliferation, and maturation, and ultimately whether rejection or tolerance are induced. This novel hypothesis integrates many receptor-ligand and cell-cell interactions, and places these in the context of secondary lymphoid organ structure. Our currently funded investigations are focused on the fate and differentiation of Treg, T cell migration and trafficking, and the role of T-pDC interactions. While T cell responses have a central role in transplantation tolerance, our new preliminary data suggest novel and important roles in the LN for B cells, stromal cells, stromal fibers, and other LN elements in the clustered interaction that leads to tolerization. To investigate the role of these novel cellular and structural elements in LN clustered interactions, we propose the following specific aims: Specific Aim 1. Determine the roles of B cells in LN structure in tolerance using a transplant model that allows the specific tracking of antigen specific T cells and specific alloantigen presenting antigen presenting cells (ARC), we will investigate the roles of B cells in the LN during the T-APC clustered interaction that results in tolerization. Analyses will focus on B cell ARC function, chemokine production, and immunoglobulin production. Specific Aim 2. Determine the LN stromal structures and cells essential for tolerance We will investigate the role of stromal cells and stromal cell derived elements in the LN in the T-APC clustered interaction during tolerization. Specific Aim 3. Determine the role of invariant innate cellular immune responses in tolerance We will determine how other cellular elements, that respond to innate and invariant signals, affect the T-APC clustered interaction, LN structure, and tolerization.